Preparation and application of fluorescent monoclonal antibodies recognizing goat CD4+CD25+ regulatory T cells.

Regulatory T cells (Tregs) are a subset of T cells participating in a variety of diseases including mycoplasmal pneumonia, contagious ecthyma, and so on. The role of Tregs in goat contagious ecthyma is not completely understood due to the lack of species-specific antibodies. Here, we developed a combination of CD4 and CD25 fluorescence monoclonal antibodies (mAb) to recognize goat Tregs and assessed its utility in flow cytometry, immunofluorescence staining. Using immunofluorescence staining, we found that the frequency of Treg cells was positively correlated with the viral load during orf virus infection. These antibodies could serve as important tools to monitor Tregs during orf virus infection in goats. KEY POINTS: • A combination of fluorescent mAbs (C11 and D12) was prepared for the detection of goat Tregs. • C11 and D12 are effective in flow cytometry, immunofluorescence staining, and C11 has excellent species specificity. • The frequency of Treg cells was positively correlated with the viral load during orf virus infection.

The CD4 transmembrane GGXXG and juxtamembrane (C/F)CV+C motifs mediate pMHCII-specific signaling independently of CD4-LCK interactions.

CD4+ T cell activation is driven by five-module receptor complexes. The T cell receptor (TCR) is the receptor module that binds composite surfaces of peptide antigens embedded within MHCII molecules (pMHCII). It associates with three signaling modules (CD3γε, CD3δε, and CD3ζζ) to form TCR-CD3 complexes. CD4 is the coreceptor module. It reciprocally associates with TCR-CD3-pMHCII assemblies on the outside of a CD4+ T cells and with the Src kinase, LCK, on the inside. Previously, we reported that the CD4 transmembrane GGXXG and cytoplasmic juxtamembrane (C/F)CV+C motifs found in eutherian (placental mammal) CD4 have constituent residues that evolved under purifying selection (Lee et al., 2022). Expressing mutants of these motifs together in T cell hybridomas increased CD4-LCK association but reduced CD3ζ, ZAP70, and PLCγ1 phosphorylation levels, as well as IL-2 production, in response to agonist pMHCII. Because these mutants preferentially localized CD4-LCK pairs to non-raft membrane fractions, one explanation for our results was that they impaired proximal signaling by sequestering LCK away from TCR-CD3. An alternative hypothesis is that the mutations directly impacted signaling because the motifs normally play an LCK-independent role in signaling. The goal of this study was to discriminate between these possibilities. Using T cell hybridomas, our results indicate that: intracellular CD4-LCK interactions are not necessary for pMHCII-specific signal initiation; the GGXXG and (C/F)CV+C motifs are key determinants of CD4-mediated pMHCII-specific signal amplification; the GGXXG and (C/F)CV+C motifs exert their functions independently of direct CD4-LCK association. These data provide a mechanistic explanation for why residues within these motifs are under purifying selection in jawed vertebrates. The results are also important to consider for biomimetic engineering of synthetic receptors.

[Clinical Characteristics of CD4-CD56+ Blastic Plasmacytoid Dendritic Cell Neoplasm].

OBJECTIVE: To explore the clinical manifestations, pathological features, immunophenotype, as well as diagnosis, treatment and prognosis of patients with CD4-CD56+ blastic plasmacytoid dendritic cell neoplasm (BPDCN), in order to further understand the rare disease. METHODS: The clinical data, laboratory examinations and treatment regimens of two patients with CD4-CD56+ BPDCN in the First Affiliated Hospital of Wannan Medical College were retrospectively analyzed. RESULTS: The two patients were both elderly males with tumor involved in skin, bone marrow, lymph nodes, etc. Immunohistochemical results of skin lesions showed that both CD56 and CD123 were positive, while CD4, CD34, TdT, CD3, CD20, MPO and EBER were negative. Flow cytometry of bone marrow demonstrated that CD56, CD123, and CD304 were all positive, while specific immune markers of myeloid and lymphoid were negative. Two patients were initially very sensitive to acute lymphoblastic leukemia or lymphomatoid chemotherapy regimens, but prone to rapid relapse. The overall survival of both patients was 36 months and 4 months, respectively. CONCLUSION: CD4-CD56+ BPDCN is very rare and easily misdiagnosed as other hematological tumors with poor prognosis. Acute lymphoblastic leukemia or lymphomatoid therapy should be used first to improve the poor prognosis.

[Circulating populations of CD4+ CD25+ CD127- regulatory t lymphocytes in peripheral blood of allergic asthmatic children]. / Determinación de poblaciones circulantes en sangre periférica de linfocitos T, reguladores CD4+ CD25+ CD127-, en niños asmáticos alérgicos.

OBJECTIVE: To carry out a preliminary analysis on the Treg lymphocyte counts present in the peripheral blood of allergic asthmatic children from the city of Cartagena, Colombia, compared to healthy controls. METHODS: We compared cytometry counts of ten asthmatic patients (age 7-16 years) and seven healthy controls (6-12 years), recruited in the city of Cartagena. Peripheral blood samples were stained using Cytek's 14-color cFluor Immunoprofiling kit (Cytek® cFluor® Immunoprofiling Kit 14 Color RUO kit), and analyzed on a Northern Lights™ spectral cytometer (Cytek® Biosciences, Fremont, CA, USA), to read 50.000 events per sample. The data obtained were analyzed in SpectroFlo® and FlowJo. The study was approved by the ethics committee of the University of Cartagena (SGR, Grant BPIN2020000100405). RESULTS: The frequency of CD3+, CD4+, CD25+, CD127- Tregs was 11% of all CD4+ T cells, with a range of minimum 8,1% and maximum 17,7%. There was no significant difference in the proportion of Tregs between allergic asthmatic patients and healthy controls (P = 0,2). CONCLUSIONS: With this preliminary sample size, no significant differences were found in the Treg lymphocyte population between allergic asthmatic patients and healthy controls. The 14-color multiplexed panel is a useful tool not only to count CD3+ and CD4+ populations, but also to obtain the percentage of regulatory T cells using cell surface markers.

OBJETIVO: Realizar un análisis preliminar sobre los conteos de linfocitos Tregs presentes en sangre periférica de niños asmáticos alérgicos de la ciudad de Cartagena, comparado con controles sanos. MÉTODOS: Se compararon los conteos de citometría de diez pacientes asmáticos (entre 7 y16 años) y siete controles sanos (entre 6 y12 años), reclutados en la ciudad de Cartagena. La muestra de sangre periférica fue teñida empleando el kit de inmunofenotipo multiplexado de 14 colores de Cytek (Cytek® cFluor® Immunoprofiling Kit 14 Color), y analizada en un citómetro espectral Northern Lights™ (Cytek® Biosciences, Fremont, CA, USA), a lectura de 50.000 eventos por muestra. Los datos obtenidos fueron analizados en SpectroFlo® y FlowJo. El estudio fue aprobado por el Comité de Ética de la Universidad de Cartagena. RESULTADOS: El panel de tinción funcionó apropiadamente y dentro de los parámetros apropiados. Se obtuvo un promedio de células Tregs CD3+, CD4+, CD25+ y CD127- del 11% de todos los CD4+ en las muestras estudiadas, con un rango de mínimo de 8,1% y un máximo de 17,7%. No hubo diferencias significativas en la proporción de linfocitos Tregs entre los pacientes asmáticos alérgicos y los controles sanos (P = 0.2). CONCLUSIONES: Con este tamaño de muestra preliminar, no se encontraron diferencias significativas en la población de linfocitos Tregs entre los pacientes asmáticos alérgicos y los controles sanos. El panel multiplexado de 14 colores es una herramienta útil no solo para derivar las poblaciones CD3+ y CD4+, sino también para obtener el porcentaje de células T reguladoras empleando marcadores de superficie celular.

Stoichiometry for entry and binding properties of the Env protein of R5 T cell-tropic HIV-1 and its evolutionary variant of macrophage-tropic HIV-1.

Human immunodeficiency virus type 1 typically requires a high density of CD4 for efficient entry as a mechanism to target CD4+ T cells (T-tropic), with CCR5 being used most often as the coreceptor. When target T cells are limiting, the virus can evolve to infect cells with a low density of CD4 such as macrophages (M-tropic). The entry phenotype is known to be encoded in the viral Env protein on the surface of the virus particle. Using data showing a dose response for infectivity based on CD4 surface density, we built a model consistent with T-tropic viruses requiring multiple CD4 molecules to mediate infection, whereas M-tropic viruses can infect cells using a single CD4 receptor molecule interaction. We also found that T-tropic viruses bound to the surface of cells with a low density of CD4 are released more slowly than M-tropic viruses which we modeled to be due to multiple interactions of the T-tropic virus with multiple CD4 molecules to allow the initial stable binding. Finally, we found that some M-tropic Env proteins, as the gp120 subunit, possess an enhanced affinity for CD4 compared with their T-tropic pair, indicating that the evolution of macrophage tropism can be reflected both in the closed Env trimer conformation on the virion surface and, in some cases, also in the open confirmation of gp120 Env. Collectively, these studies reveal differences in the stoichiometry of interaction of T-tropic and M-tropic viruses with CD4 and start to identify the basis of binding differences at the biochemical level. IMPORTANCE: Human immunodeficiency virus type 1 normally targets CD4+ T cells for viral replication. When T cells are limiting, the virus can evolve to infect myeloid cells. The evolutionary step involves a change from requiring a high surface density of CD4 for entry to being able to infect cells with a low density of CD4, as is found on myeloid lineage cells such as macrophage and microglia. Viruses able to infect macrophages efficiently are most often found in the CNS late in the disease course, and such viruses may contribute to neurocognitive impairment. Here, we examine the CD4 binding properties of the viral Env protein to explore these two different entry phenotypes.

High-throughput virtual search of small molecules for controlling the mechanical stability of human CD4.

Protein mechanical stability determines the function of a myriad of proteins, especially proteins from the extracellular matrix. Failure to maintain protein mechanical stability may result in diseases and disorders such as cancer, cardiomyopathies, or muscular dystrophy. Thus, developing mutation-free approaches to enhance and control the mechanical stability of proteins using pharmacology-based methods may have important implications in drug development and discovery. Here, we present the first approach that employs computational high-throughput virtual screening and molecular docking to search for small molecules in chemical libraries that function as mechano-regulators of the stability of human cluster of differentiation 4, receptor of HIV-1. Using single-molecule force spectroscopy, we prove that these small molecules can increase the mechanical stability of CD4D1D2 domains over 4-fold in addition to modifying the mechanical unfolding pathways. Our experiments demonstrate that chemical libraries are a source of mechanoactive molecules and that drug discovery approaches provide the foundation of a new type of molecular function, that is, mechano-regulation, paving the way toward mechanopharmacology.

A VRC13-like bNAb response is associated with complex escape pathways in HIV-1 envelope.

The rational design of HIV-1 immunogens to trigger the development of broadly neutralizing antibodies (bNAbs) requires understanding the viral evolutionary pathways influencing this process. An acute HIV-1-infected individual exhibiting >50% plasma neutralization breadth developed neutralizing antibody specificities against the CD4-binding site (CD4bs) and V1V2 regions of Env gp120. Comparison of pseudoviruses derived from early and late autologous env sequences demonstrated the development of >2 log resistance to VRC13 but not to other CD4bs-specific bNAbs. Mapping studies indicated that the V3 and CD4-binding loops of Env gp120 contributed significantly to developing resistance to the autologous neutralizing response and that the CD4-binding loop (CD4BL) specifically was responsible for the developing resistance to VRC13. Tracking viral evolution during the development of this cross-neutralizing CD4bs response identified amino acid substitutions arising at only 4 of 11 known VRC13 contact sites (K282, T283, K421, and V471). However, each of these mutations was external to the V3 and CD4BL regions conferring resistance to VRC13 and was transient in nature. Rather, complete resistance to VRC13 was achieved through the cooperative expression of a cluster of single amino acid changes within and immediately adjacent to the CD4BL, including a T359I substitution, exchange of a potential N-linked glycosylation (PNLG) site to residue S362 from N363, and a P369L substitution. Collectively, our data characterize complex HIV-1 env evolution in an individual developing resistance to a VRC13-like neutralizing antibody response and identify novel VRC13-associated escape mutations that may be important to inducing VRC13-like bNAbs for lineage-based immunogens.IMPORTANCEThe pursuit of eliciting broadly neutralizing antibodies (bNAbs) through vaccination and their use as therapeutics remains a significant focus in the effort to eradicate HIV-1. Key to our understanding of this approach is a more extensive understanding of bNAb contact sites and susceptible escape mutations in HIV-1 envelope (env). We identified a broad neutralizer exhibiting VRC13-like responses, a non-germline restricted class of CD4-binding site antibody distinct from the well-studied VRC01-class. Through longitudinal envelope sequencing and Env-pseudotyped neutralization assays, we characterized a complex escape pathway requiring the cooperative evolution of four amino acid changes to confer complete resistance to VRC13. This suggests that VRC13-class bNAbs may be refractory to rapid escape and attractive for therapeutic applications. Furthermore, the identification of longitudinal viral changes concomitant with the development of neutralization breadth may help identify the viral intermediates needed for the maturation of VRC13-like responses and the design of lineage-based immunogens.

The relationship between high ratios of CD4/FOXP3 and CD8/CD163 and the improved survivability of metastatic triple-negative breast cancer patients: a multicenter cohort study.

BACKGROUND: Triple-negative breast cancer (TNBC) has been documented as the most aggressive subtype of breast cancer. This study aimed to analyze antitumor and protumor immune activities, and their ratios as significant prognostic biomarkers in metastatic TNBC (mTNBC). METHODS: A multicenter cohort study was conducted among 103 de novo mTNBC patients. The expression of CD8 and CD163 was evaluated using immunohistochemistry staining, CD4 and FOXP3 using double-staining immunohistochemistry, and PD-L1 using immunohistochemistry and RT-PCR. RESULTS: Multivariate analysis revealed that high CD4/FOXP3 (HR 1.857; 95% CI 1.049-3.288; p = 0.034) and the CD8/CD163 ratio (HR 2.089; 95% CI 1.174-3.717; p = 0.012) yield significantly improved 1 year overall survival (OS). Kaplan-Meier analysis showed that high levels of CD4 (p = 0.023), CD8 (p = 0.043), CD4/FOXP3 (p = 0.016), CD8/FOXP3 (p = 0.005), CD8/CD163 (p = 0.005) ratios were significantly associated with higher rate of 1 year OS. Furthermore, 1 year OS was directly correlated with antitumor CD4 (R = 0.233; p = 0.018) and CD8 (R = 0.219; p = 0.026) and was indirectly correlated with protumor CD163 and FOXP3 through CD4/FOXP3 (R = 0.282; p = 0.006), CD4/CD163 (R = 0.239; p = 0.015), CD8/FOXP3 (R = 0.260; p = 0.008), and CD8/CD163 (R = 0.258; p = 0.009). CONCLUSION: This is the first study to demonstrate that high levels of CD4/FOXP3 and CD8/CD163 significantly improved the 1 year OS in de novo mTNBC patients. Thus, we recommend the application of these markers as prognosis determination and individual treatment decision.

Vaccine induction of CD4-mimicking HIV-1 broadly neutralizing antibody precursors in macaques.

The CD4-binding site (CD4bs) is a conserved epitope on HIV-1 envelope (Env) that can be targeted by protective broadly neutralizing antibodies (bnAbs). HIV-1 vaccines have not elicited CD4bs bnAbs for many reasons, including the occlusion of CD4bs by glycans, expansion of appropriate naive B cells with immunogens, and selection of functional antibody mutations. Here, we demonstrate that immunization of macaques with a CD4bs-targeting immunogen elicits neutralizing bnAb precursors with structural and genetic features of CD4-mimicking bnAbs. Structures of the CD4bs nAb bound to HIV-1 Env demonstrated binding angles and heavy-chain interactions characteristic of all known human CD4-mimicking bnAbs. Macaque nAb were derived from variable and joining gene segments orthologous to the genes of human VH1-46-class bnAb. This vaccine study initiated in primates the B cells from which CD4bs bnAbs can derive, accomplishing the key first step in the development of an effective HIV-1 vaccine.

Implications of the 375W mutation for HIV-1 tropism and vaccine development.

Understanding how different amino acids affect the HIV-1 envelope (Env) trimer will greatly help the design and development of vaccines that induce broadly neutralizing antibodies (bnAbs). A tryptophan residue at position 375 that opens the CD4 binding site without modifying the trimer apex was identified using our saturation mutagenesis strategy. 375W was introduced into a large panel of 27 transmitted/founder, acute stage, chronic infection, and AIDS macrophage-tropic and non-macrophage-tropic primary envelopes from different clades (A, B, C, D, and G) as well as complex and circulating recombinants. We evaluated soluble CD4 and monoclonal antibody neutralization of WT and mutant Envs together with macrophage infection. The 375W substitution increased sensitivity to soluble CD4 in all 27 Envs and macrophage infection in many Envs including an X4 variant. Importantly, 375W did not impair or abrogate neutralization by potent bnAbs. Variants that were already highly macrophage tropic were compromised for macrophage tropism, indicating that other structural factors are involved. Of note, we observed a macrophage-tropic (clade G) and intermediate macrophage-tropic (clades C and D) primary Envs from the blood and not from the central nervous system (CNS), indicating that such variants could be released from the brain or evolve outside the CNS. Our data also indicate that "intermediate" macrophage-tropic variants should belong to a new class of HIV-1 tropism. These Envs infected macrophages more efficiently than non-macrophage-tropic variants without reaching the high levels of macrophage-tropic brain variants. In summary, we show that 375W is ideal for inclusion into HIV-1 vaccines, increasing Env binding to CD4 for widely diverse Envs from different clades and disease stages.IMPORTANCESubstitutions exposing the CD4 binding site (CD4bs) on HIV-1 trimers but still occluding non-neutralizing, immunogenic epitopes are desirable to develop HIV-1 vaccines. If such substitutions induce similar structural changes in trimers across diverse clades, they could be exploited for the development of multi-clade envelope (Env) vaccines. We show that the 375W substitution increases CD4 affinity for envelopes of all clades, circulating recombinant forms, and complex Envs tested, independent of disease stage. Clade B and C Envs with an exposed CD4bs were described for macrophage-tropic strains from the central nervous system (CNS). Here, we show that intermediate (clades C and D) and macrophage-tropic (clade G) envelopes can be detected outside the CNS. Vaccines targeting the CD4bs will be particularly effective against such strains and CNS disease.

Inactivation of cell-free HIV-1 by designing potent peptides based on mutations in the CD4 binding site.

Human immunodeficiency virus type 1 (HIV-1) is a major global health problem, with over 38 million people infected worldwide. Current anti-HIV-1 drugs are limited in their ability to prevent the virus from replicating inside host cells, making them less effective as preventive measures. In contrast, viral inhibitors that inactivate the virus before it can bind to a host cell have great potential as drugs. In this study, we aimed to design mutant peptides that could block the interaction between gp120 and the CD4 receptor on host cells, thus preventing HIV-1 infection. We designed a 20-amino-acid peptide that mimicked the amino acids of the CD4 binding site and docked it to gp120. Molecular dynamics simulations were performed to calculate the energy of MMPBSA (Poisson-Boltzmann Surface Area) for each residue of the peptide, and unfavorable energy residues were identified as potential mutation points. Using MAESTRO (Multi AgEnt STability pRedictiOn), we measured ΔΔG (change in the change in Gibbs free energy) for mutations and generated a library of 240 mutated peptides using OSPREY software. The peptides were then screened for allergenicity and binding affinity. Finally, molecular dynamics simulations (via GROMACS 2020.2) and control docking (via HADDOCK 2.4) were used to evaluate the ability of four selected peptides to inhibit HIV-1 infection. Three peptides, P3 (AHRQIRQWFLTRGPNRSLWQ), P4 (VHRQIRQWFLTRGPNRSLWQ), and P9 (AHRQIRQMFLTRGPNRSLWQ), showed practical and potential as HIV inhibitors, based on their binding affinity and ability to inhibit infection. These peptides have the ability to inactivate the virus before it can bind to a host cell, thus representing a promising approach to HIV-1 prevention. Our findings suggest that mutant peptides designed to block the interaction between gp120 and the CD4 receptor have potential as HIV-1 inhibitors. These peptides could be used as preventive measures against HIV-1 transmission, and further research is needed to evaluate their safety and efficacy in clinical settings.

Idiotopes of antibodies against HIV-1 CD4-induced epitope shared with those against microorganisms.

Induction of antibodies (Abs) against the conformational CD4-induced (CD4i) epitope is frequent in HIV-1 infection. However, the mechanism of development of anti-CD4i Abs is unclear. We used anti-idiotypic (aID) monoclonal Abs (mAbs) of anti-CD4i mAbs to isolate anti-CD4i mAbs from infected subjects and track the causative antigens. One anti-aID mAb sorted from infected subjects by aID mAbs had the characteristics of anti-CD4i Abs, including IGHV1-69 usage and ability to bind to HIV-1 Env enhanced by sCD4. Critical amino acid sequences for the binding of six anti-aID mAbs, with shared idiotope to anti-CD4i mAbs, were analysed by phage display. The identified amino acid sequences showed similarity to proteins from human microbiota and infectious agents. Peptides synthesized from Caudoviricetes sp and Vibrio vulnificus based on the identified sequences were reactive to most anti-aID and some anti-CD4i mAbs. These results suggest that anti-CD4i Abs may evolve from B cells primed by microorganisms.

A combined biochemical and cellular approach reveals Zn2+-dependent hetero- and homodimeric CD4 and Lck assemblies in T cells.

The CD4 or CD8 co-receptors' interaction with the protein-tyrosine kinase Lck initiates the tyrosine phosphorylation cascade leading to T cell activation. A critical question is: to what extent are co-receptors and Lck coupled? Our contribution concerns Zn2+, indispensable for CD4- and CD8-Lck formation. We combined biochemical and cellular approaches to show that dynamic fluctuations of free Zn2+ in physiological ranges influence Zn(CD4)2 and Zn(CD4)(Lck) species formation and their ratio, although the same Zn(Cys)2(Cys)2 cores. Moreover, we demonstrated that the affinity of Zn2+ to CD4 and CD4-Lck species differs significantly. Increased intracellular free Zn2+ concentration in T cells causes higher CD4 partitioning in the plasma membrane. We additionally found that CD4 palmitoylation decreases the specificity of CD4-Lck formation in the reconstituted membrane model. Our findings help elucidate co-receptor-Lck coupling stoichiometry and demonstrate that intracellular free Zn2+ has a major role in the interplay between CD4 dimers and CD4-Lck assembly.

The V2 domain of HIV gp120 mimics an interaction between CD4 and integrin ⍺4ß7.

The CD4 receptor, by stabilizing TCR-MHC II interactions, plays a central role in adaptive immunity. It also serves as the HIV docking receptor. The HIV gp120 envelope protein binds directly to CD4. This interaction is a prerequisite for viral entry. gp120 also binds to ⍺4ß7, an integrin that is expressed on a subset of memory CD4+ T cells. HIV tropisms for CD4+ T cells and gut tissues are central features of HIV pathogenesis. We report that CD4 binds directly to ⍺4ß7 in a dynamic way, consistent with a cis regulatory interaction. The molecular details of this interaction are related to the way in which gp120 interacts with both receptors. Like MAdCAM-1 and VCAM-1, two recognized ligands of ⍺4ß7, the binding interface on CD4 includes 2 sites (1° and accessory), distributed across its two N-terminal IgSF domains (D1 and D2). The 1° site includes a sequence in the G ß-strand of CD4 D2, KIDIV, that binds directly to ⍺4ß7. This pentapeptide sequence occurs infrequently in eukaryotic proteins. However, a closely related and conserved sequence, KLDIV, appears in the V2 domain of gp120. KLDIV mediates gp120-⍺4ß7 binding. The accessory ⍺4ß7 binding site on CD4 includes Phe43. The Phe43 aromatic ring protrudes outward from one edge of a loop connecting the C'C" strands of CD4 D1. Phe43 is a principal contact for HIV gp120. It interacts with conserved residues in the recessed CD4 binding pocket. Substitution of Phe43 abrogates CD4 binding to both gp120 and ⍺4ß7. As such, the interactions of gp120 with both CD4 and ⍺4ß7 reflect elements of their interactions with each other. These findings indicate that gp120 specificities for CD4 and ⍺4ß7 are interrelated and suggest that selective pressures which produced a CD4 tropic virus that replicates in gut tissues are linked to a dynamic interaction between these two receptors.

Potent latency reversal by Tat RNA-containing nanoparticle enables multi-omic analysis of the HIV-1 reservoir.

The development of latency reversing agents that potently reactivate HIV without inducing global T cell activation would benefit the field of HIV reservoir research and could pave the way to a functional cure. Here, we explore the reactivation capacity of a lipid nanoparticle containing Tat mRNA (Tat-LNP) in CD4 T cells from people living with HIV undergoing antiretroviral therapy (ART). When combined with panobinostat, Tat-LNP induces latency reversal in a significantly higher proportion of latently infected cells compared to PMA/ionomycin (≈ 4-fold higher). We demonstrate that Tat-LNP does not alter the transcriptome of CD4 T cells, enabling the characterization of latently infected cells in their near-native state. Upon latency reversal, we identify transcriptomic differences between infected cells carrying an inducible provirus and non-infected cells (e.g. LINC02964, GZMA, CCL5). We confirm the transcriptomic differences at the protein level and provide evidence that the long non-coding RNA LINC02964 plays a role in active HIV infection. Furthermore, p24+ cells exhibit heightened PI3K/Akt signaling, along with downregulation of protein translation, suggesting that HIV-infected cells display distinct signatures facilitating their long-term persistence. Tat-LNP represents a valuable research tool for in vitro reservoir studies as it greatly facilitates the in-depth characterization of HIV reservoir cells' transcriptome and proteome profiles.

TScan-II: A genome-scale platform for the de novo identification of CD4+ T cell epitopes.

CD4+ T cells play fundamental roles in orchestrating immune responses and tissue homeostasis. However, our inability to associate peptide human leukocyte antigen class-II (HLA-II) complexes with their cognate T cell receptors (TCRs) in an unbiased manner has hampered our understanding of CD4+ T cell function and role in pathologies. Here, we introduce TScan-II, a highly sensitive genome-scale CD4+ antigen discovery platform. This platform seamlessly integrates the endogenous HLA-II antigen-processing machinery in synthetic antigen-presenting cells and TCR signaling in T cells, enabling the simultaneous screening of multiple HLAs and TCRs. Leveraging genome-scale human, virome, and epitope mutagenesis libraries, TScan-II facilitates de novo antigen discovery and deep exploration of TCR specificity. We demonstrate TScan-II's potential for basic and translational research by identifying a non-canonical antigen for a cancer-reactive CD4+ T cell clone. Additionally, we identified two antigens for clonally expanded CD4+ T cells in Sjögren's disease, which bind distinct HLAs and are expressed in HLA-II-positive ductal cells within affected salivary glands.

Single cell and spatial transcriptomics analysis of kidney double negative T lymphocytes in normal and ischemic mouse kidneys.

T cells are important in the pathogenesis of acute kidney injury (AKI), and TCR+CD4-CD8- (double negative-DN) are T cells that have regulatory properties. However, there is limited information on DN T cells compared to traditional CD4+ and CD8+ cells. To elucidate the molecular signature and spatial dynamics of DN T cells during AKI, we performed single-cell RNA sequencing (scRNA-seq) on sorted murine DN, CD4+, and CD8+ cells combined with spatial transcriptomic profiling of normal and post AKI mouse kidneys. scRNA-seq revealed distinct transcriptional profiles for DN, CD4+, and CD8+ T cells of mouse kidneys with enrichment of Kcnq5, Klrb1c, Fcer1g, and Klre1 expression in DN T cells compared to CD4+ and CD8+ T cells in normal kidney tissue. We validated the expression of these four genes in mouse kidney DN, CD4+ and CD8+ T cells using RT-PCR and Kcnq5, Klrb1, and Fcer1g genes with the NIH human kidney precision medicine project (KPMP). Spatial transcriptomics in normal and ischemic mouse kidney tissue showed a localized cluster of T cells in the outer medulla expressing DN T cell genes including Fcer1g. These results provide a template for future studies in DN T as well as CD4+ and CD8+ cells in normal and diseased kidneys.

HIV-1 Env trimers asymmetrically engage CD4 receptors in membranes.

Human immunodeficiency virus 1 (HIV-1) infection is initiated by binding of the viral envelope glycoprotein (Env) to the cell-surface receptor CD41-4. Although high-resolution structures of Env in a complex with the soluble domains of CD4 have been determined, the binding process is less understood in native membranes5-13. Here we used cryo-electron tomography to monitor Env-CD4 interactions at the membrane-membrane interfaces formed between HIV-1 and CD4-presenting virus-like particles. Env-CD4 complexes organized into clusters and rings, bringing the opposing membranes closer together. Env-CD4 clustering was dependent on capsid maturation. Subtomogram averaging and classification revealed that Env bound to one, two and finally three CD4 molecules, after which Env adopted an open state. Our data indicate that asymmetric HIV-1 Env trimers bound to one and two CD4 molecules are detectable intermediates during virus binding to host cell membranes, which probably has consequences for antibody-mediated immune responses and vaccine immunogen design.

Intermediate conformations of CD4-bound HIV-1 Env heterotrimers.

HIV-1 envelope (Env) exhibits distinct conformational changes in response to host receptor (CD4) engagement. Env, a trimer of gp120 and gp41 heterodimers, has been structurally characterized in a closed, prefusion conformation with closely associated gp120s and coreceptor binding sites on gp120 V3 hidden by V1V2 loops1-4 and in fully saturated CD4-bound open Env conformations with changes including outwardly rotated gp120s and displaced V1V2 loops3-9. To investigate changes resulting from substoichiometric CD4 binding, we solved single-particle cryo-electron microscopy (cryo-EM) structures of soluble, native-like heterotrimeric Envs bound to one or two CD4 molecules. Most of the Env trimers bound to one CD4 adopted the closed, prefusion Env state, with a minority exhibiting a heterogeneous partially open Env conformation. When bound to two CD4s, the CD4-bound gp120s exhibited an open Env conformation including a four-stranded gp120 bridging sheet and displaced gp120 V1V2 loops that expose the coreceptor sites on V3. The third gp120 adopted an intermediate, occluded-open state10 that showed gp120 outward rotation but maintained the prefusion three-stranded gp120 bridging sheet with only partial V1V2 displacement and V3 exposure. We conclude that most of the engagements with one CD4 molecule were insufficient to stimulate CD4-induced conformational changes, whereas binding two CD4 molecules led to Env opening in CD4-bound protomers only. The substoichiometric CD4-bound soluble Env heterotrimer structures resembled counterparts derived from a cryo-electron tomography study of complexes between virion-bound Envs and membrane-anchored CD4 (ref. 11), validating their physiological relevance. Together, these results illuminate intermediate conformations of HIV-1 Env and illustrate its structural plasticity.

Association of Alcohol Consumption With CD4 Recovery After Antiretroviral Therapy Initiation in St. Petersburg, Russia.

BACKGROUND: Delayed CD4 recovery after initiating antiretroviral therapy (ART) is a novel potential mechanism by which alcohol consumption leads to increased morbidity and mortality in people with HIV. We hypothesized that alcohol consumption at ART initiation is associated with slower CD4 recovery. METHODS: We retrospectively analyzed 2 pooled longitudinal alcohol/HIV cohorts (2014-2019) in St. Petersburg, Russia. Eligible participants initiated the first ART during parent studies; had alcohol consumption assessed by the blood biomarker, phosphatidylethanol (PEth), at the last research visit before ART initiation; and had ≥1 CD4 count measurement before and after initiating ART. Participants were stratified by low, moderate, and high PEth (<8, 8-80, and >80 ng/mL, respectively). We used random-effects piecewise linear regression models to estimate CD4 recovery, defined as CD4 count change per 30 days after ART initiation, by the alcohol group. RESULTS: Of 60 eligible participants, median age was 34 years and 28% were female. The median pre-ART PEth in the low, moderate, and high PEth groups were <8, 23, and 232 ng/mL, respectively. After starting ART, the CD4 count increased by 13.60 cells/mm3/mo (95% CI: 0.33 to 26.87) with low PEth, 0.93 cells/mm3/mo (95% CI: -6.18 to 8.04) with moderate PEth, and 2.33 cells/mm3/mo (95% CI: -3.44 to 8.09) with high PEth. CONCLUSIONS: Among Russians with HIV, we observed faster CD4 recovery after ART initiation in those with low alcohol consumption compared with those with moderate and high alcohol consumption, as assessed by PEth. This analysis provides further evidence for the possible value of alcohol reduction interventions for people with HIV who are initiating ART.